Control of phosphate starvation responses in Arabidopsis thaliana: new regulators and regulatory interactions

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Faculta de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 25-11-2014To cope with growth under Pi starvation conditions, plants have evolved a series of morphological and biochemical adaptations aimed to survive at best the stress situation. Pi starvation signaling mechanism in plants has been widely studied in the past two decades. However, there were some gaps in the knowledge of this pathway; for instance, on the mechanism of regulation of PHR1 (master regulator of Pi starvation responses) activity, on the properties of the Pi sensor, the complete TF set controlling the transcriptional networks underlying Pi ion homeostasis etc,. In this study we have contributed to the knowledge of phosphate starvation signaling at 3 different fronts. 1) Identification of candidate TFs controlling PSRs using ionomics -we used a large scale ionomic profiling approach to study the elemental profile of the transgenic lines of TRANSPLANTA collection conditionally overexpressing TFs. In general, we observed that alterations in the ionome involved disturbances in the levels of many elements. Giving emphasis to P nutrient signaling, we selected 5 TF candidates (belonging to families of DREB, bZIP, NAC and KNAT) whose ionomic pattern indicated potential correlations between P and other elements like Zn, Fe and Mn. 2) SPX1 is a Pi dependent inhibitor of PHR1. Following a yeast two hybrid approach, we identified SPX1 as an interactor of PHR1. Subsequent characterization studies included physiological and transcriptomic analysis of spx1spx2 mutants, Coimmunoprecipitation assay in-planta and in-vitro, as well as DNA binding assays. As a result of this characterization, we established that SPX1 is a Pi dependent inhibitor of PHR1, qualifying it as a sensor component. 3) New roles of PHO2 and NLA in Pi starvation signaling. In this study, we found that PHO2 and NLA interact with each other suggesting they act in concert in the ubiquitination pathway. In line with the previous finding that the negative growth regulators bHLH149 is a target of PHO2, bHLH149 is also shown to be regulated by NLA, reinforcing the link between Pi starvation signaling and growth control. In addition, SPX1 is also shown to be a PHO2/NLA target, contributing to form a negative regulatory loop in Pi starvation signaling involving PHR1, NLA, PHO2 and SPX1.Las plantas han desarrollado una serie de respuestas morfológicas y bioquímicas destinadas a adaptar su crecimiento en condiciones de bajo Pi, en el suelo. El mecanismo de señalización de ayuno de fosfato en plantas ha sido ampliamente estudiado en las últimas dos décadas. Sin embargo, todavía existían importantes lagunas en el conocimiento de esta ruta; por ejemplo, sobre cómo se regula la actividad de PHR1 (regulador maestro de las respuestas al ayuno de Pi) ; sobre la naturaleza y el modo de accion del sensor de Pi y sobre el conjunto de TFs de las redes de transcripción subyacentes a la homeostasis de Pi etc,. En este estudio hemos contribuido en tres aspectos diferentes al conocimiento de la señalización del ayuno de fosfato. 1) Identificación de nuevos TFs candidatos mediante aproximaciones ionómicas. – Se ha realizado un analisis a gran escala del pefil ionómico de lineas TRANSPLANTA que sobreexpresan condicionalmente TFs de Arabidopsis, para detectar TFs cuya sobreexpresión altera el ionoma. En los casos encontrados, en general, se observó que las alteraciones en el ionoma implican alteraciones en los niveles de muchos elementos. Centrándonos en los TFs relacionados con la homeostasis de Pi, se seleccionaron 5 TF candidatos (pertenecientes a familias de DREB, bZIP, NAC y KNAT) cuyo patrón ionómico refleja las posibles correlaciones entre P y otros elementos como Zn, Fe y Mn. 2) SPX1 es un inhibidor de PHR1 dependiente de Pi - Siguiendo una aproximación basada en el método de los dos híbridos de levadura, se identificó SPX1 como un interactor de PHR1. Estudios de caracterización posteriores incluyeron análisis fisiológicos y transcriptómicos de mutantes spx1spx2, ensayos de co-inmunoprecipitación in planta e in vitro, así como ensayos de unión de ADN. Como resultado de esta caracterización, se estableció que SPX1 es un inhibidor PHR1 directamente dependiente de Pi, cualificándolo como un componente del sensor de Pi. 3) Nuevas funciones de PHO2 y NLA en la vía de señalización de ayuno de Pi. En este estudio, se encontró que PHO2 y NLA interaccionan entre sí lo que sugiere que actúan en concierto en la ruta de ubiquitinación implicada en la señalización de Pi. En línea con datos previos que establecieron que el inhibidor de crecimiento bHlH149, está controlado por PH02, se ha comprobado que también está regulado por NLA, reforzando el vínculo entre la señalización del ayuno de Pi y el control del crecimiento. Además, también hemos demostrado que SPX1 es diana de PHO2 y NLA, lo que contribuye a formar un bucle regulador negativo en la señalización del ayuno de Pi que implica a PHR1, NLA, PHO2 y SPX1

The Potyviridae P1a leader protease contributes to host range specificity

[EN] The P1a protein of the ipomovirus Cucumber vein yellowing virus is one of the self-cleavage serine proteases present in Potyviridae family members. P1a is located at the N-terminal end of the viral polyprotein, and is closely related to potyviral P1 protease. For its proteolytic activity, P1a requires a still unknown host factor; this might be linked to involvement in host specificity. Here we built a series of constructs and chimeric viruses to help elucidate the role of P1a cleavage in host range definition. We demonstrate that host-dependent separation of P1a from the remainder of the polyprotein is essential for suppressing RNA silencing defenses and for efficient viral infection. Intergenus replacement of leader proteases could broaden host range definition, as shown by the local infection capacity of viral chimeras. These findings support the role of viral proteases as important determinants in host adaptation.H.S. is supported by the China Scholarship Council; F.P. and C.R. were financed by La Caixa PhD fellowships, and C.C. by a scholarship from the Spanish government (MAEC-AECID). This work was funded by Grants BIO2013-49053-R and Plant-KBBE PCIN-2013-056 from the Spanish Ministerio de Economia y Competitividad.Shan, H.; Pasin, F.; Valli, A.; Castillo, C.; Rajulu, C.; Carbonell, A.; Simon-Mateo, C.... (2015). The Potyviridae P1a leader protease contributes to host range specificity. Virology. 476:264-270. https://doi.org/10.1016/j.virol.2014.12.013S264270476ADAMS, M. J., ANTONIW, J. F., & BEAUDOIN, F. (2005). Overview and analysis of the polyprotein cleavage sites in the family Potyviridae. Molecular Plant Pathology, 6(4), 471-487. doi:10.1111/j.1364-3703.2005.00296.xBrigneti, G. (1998). Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. 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ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis.

International audiencePrior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life